Porcine IgG Research Articles

Dual-Mechanism-Driven Ratiometric Electrochemiluminescent Biosensor for Methicillin-Resistant Staphylococcus aureus.

Design of a ratiometric method is a promising pathway to improve the sensitivity and reliability of electrochemiluminescent (ECL) assay, for which the signals produced at two distinct potentials change reversely as it is applied to the target analyte. Herein, a biosensor for ECL assay of methicillin-resistant Staphylococcus aureus (MRSA) was constructed by immobilizing porcine IgG for capturing MRSA onto an electrode that was precoated with β-cyclodextrin-conjugated luminol nanoparticles (β-CD-Lu NPs) as an anodic luminophore. MOF PCN 224 loaded with an atomically distributed Zn element (PCN 224/Zn) was conjugated with phage recombinant cellular-binding domain (CBD) to act as a cathodic luminophore for tracing MRSA. After the formation of the sandwich complex of β-CD-Lu NPs-porcine IgG/MRSA/PCN 224/Zn-CBD on the biosensor, two ECL reactions were triggered with cyclic voltammetry. The anodic process of the β-CD-Lu NPs-H2O2 system and the cathodic process of the PCN 224/Zn-S2O82- system competed to react with reactive oxygen species (ROS) for producing ECL emission, which led to a reverse change of the two signals. Meanwhile, the overlap of the β-CD-Lu NPs emission spectrum and PCN 224/Zn absorption spectrum effectively triggered ECL resonance energy transfer between the donor (β-CD-Lu NPs) and the acceptor (PCN 224/Zn). Thus, a ratiometric ECL method was proposed for assaying MRSA with a dual-mechanism-driven mode. The detection limit for assaying MRSA is as low as 12 CFU/mL. The biosensor was applied to assay MRSA in various biological samples with recoveries ranging from 84.9 to 111.3%.

A nanobody inhibiting porcine reproductive and respiratory syndrome virus replication via blocking self-interaction of viral nucleocapsid protein.

Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.

PEGylated Ni Single-Atom Catalysts as Ultrasensitive Electrochemiluminescent Probes with Favorable Aqueous Dispersibility for Assaying Drug-Resistant Pathogens.

Ni single-atom catalysts (SACs) were synthesized by high-temperature calcination of nickel ions and 1,10-phenanthroline on carbon black as a carrier. Benefiting from the ultrahigh atom utilization efficiency, Ni SACs can significantly accelerate decay of dissolved oxygen to generate abundant reactive oxygen species through an oxygen reduction reaction occurring on cathodes. The generated reactive oxygen species can vastly enhance the electrochemiluminescent (ECL) signal of luminol without participation of exogenous co-reactants. To overcome the inherent unfavorable aqueous dispersibility of Ni SACs prepared by the calcination protocol, they were functionalized with highly hydrophilic PEG 2000. Thanks to the abundant carboxyl groups on PEG 2000, the PEGylated Ni SACs (Ni@PEG) can be used as ECL probes to tag biorecognition molecules. In this proof-of-principle work, an ECL biosensor for assaying methicillin-resistant Staphylococcus aureus was developed by using porcine IgG as capture molecule and phage cell-binding domain tagged with Ni@PEG as signal tracer. It shows a broad linear range of 73-7.3 × 106 CFU/mL and a low detection limit of 25 CFU/mL. The recovery values for assaying spiked samples are between 80.8 and 119.2%. It was also utilized to assess MRSA susceptibility to four antibiotics, with results consistent with those obtained by the standard broth microdilution technique. To the best of our knowledge, it is the first time to utilize aqueous dispersible SACs as highly sensitive ECL probes for developing biosensors.

Fc-Mediated Functions of Porcine IgG Subclasses.

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies.

Green fluorescent protein-fused bacteriophage cellular wall-binding domain as broad-spectrum signal probe for fluorimetry of methicillin-resistant Staphylococcus aureus strains

As a “superbug”, methicillin-resistant Staphylococcus aureus (MRSA) has long been one of the most ubiquitous drug-resistant bacteria inducing numerous nosocomial infections. To achieve effective diagnosis and following treatment decision of infectious diseases induced by MRSA, it is highly desired to establish rapid analysis and antibiotic susceptibility test methods for this pathogen. In this study, we successfully expressed a bifunctional protein by fusing green fluorescent protein and cellular wall-binding domain of bacteriophage P108. The bifunctional protein can be employed as a signal probe for broad-spectrum fluorimetry of MRSA strains because it can both bind with the target pathogen and emit intensive fluorescence. By using it as the signal probe and porcine IgG as the capture agent, MRSA can be analyzed within a dynamic range of 1.0 × 103–2.0 × 107 CFU mL−1 with a sandwich mode. The fluorimetry was also applied to test antibiotic susceptibility of this pathogen to five antibiotics, and all results are conformable with those obtained with a standard micro broth dilution method. The above results demonstrate the attractive perspective of the bifunctional protein for rapid diagnosis and effective medication of infectious diseases induced by MRSA.

Immune Characterization of a Xenogeneic Cross Circulation System for Ex Vivo Human Lung Support

<h3>Purpose</h3> Rehabilitation of injured donor lungs can reduce the shortage of organs suitable for transplantation. We previously established a platform for the maintenance and recovery of extracorporeal human lungs using cross-circulation (XC) with a porcine host. Here, we characterize the human-swine immunologic interactions in this xenogeneic system. <h3>Methods</h3> Human donor lungs declined for clinical transplantation were placed on XC with immunosuppressed (methylprednisolone, mycophenolate, tacrolimus), complement-depleted (Cobra Venom Factor) porcine hosts. Longitudinal analyses of the extracorporeal lung and porcine host were performed over 24 hours. For one of these lung (n = 1), single-cell lung suspensions and paraffin-embedded slides were prepared from biopsies of the lung collected at pre-XC baseline, 1h, and 24h. Infiltration of swine CD45+ immune cells and deposition of swine IgG and IgM in the human lung were assessed by immunohistochemistry and flow cytometry. <h3>Results</h3> The extracorporeal lung demonstrated improved PaO2/FiO2 (602%), dynamic compliance (238%), and gas exchange (O2, 974%; CO2, 198%) over the course of XC. Populations of human and porcine CD45+ leukocytes were readily identified within the post-XC lung. Human immune cells represented 1.48% and porcine immune cells represented 0.99% of isolated live single cells (Figure 1A). Infiltration of CD45+ porcine leukocytes into the lung increased over time, such that porcine neutrophils were visualized in the alveolar spaces (Figure 1B). By flow cytometry, porcine IgG was localized to lung epithelial cells (Figure 1C). Diffuse deposition of IgG and IgM was also detected by IHC (Figure 1D). <h3>Conclusion</h3> In the xenogeneic XC system, porcine immune cell and immunoglobulin infiltration of the extracorporeal human lung can be detected. Despite this, XC was sufficient to facilitate physiologic improvement in the human lung. Further work is needed to minimize cross-species immune responses during <i>ex vivo</i> organ support.

Evaluation of a Virus-like Nanoparticle Porcine Circovirus Type-2 (PCV2) Capsid Protein Fused with the Pig Immunoglobulin Fc Fragment as a Novel Vaccine Candidate against PCV2 in Mice.

Porcine circovirus Type 2 (PCV2) is a primary etiological pathogen of post-weaning multi-systemic wasting syndrome (PMWS). The capsid protein of PCV2 is the crucial immunogenic protein which can induce antibody generation and immune responses. However, there is still a lack of efficient PCV2 vaccines with high immunogenicity. In the current study, we developed a novel engineered PCV2 capsid (∆1-41aa)-pFc fusion protein (PCFP), which comprised a truncated capsid protein of PCV2 and a porcine IgG Fc fragment, fused to the capsid protein of PCV2 at the C-terminus. We found that this novel fusion protein could auto-assemble into virus-like nanoparticles with an estimated mean diameter of 22.6 nm, characterized by transmission electron microscopy. Immunization of BALB/c mice with this fusion protein significantly increased the production levels of anti-PCV2-capsid protein antibody in serum. Besides, the virus-like nanoparticles, PCFP was demonstrated to induce efficient cellular immune responses in mice, as evident by the high specific T cell reactivity to the PCFP fusion protein and the high production of the immune cytokines IFN-γ and IL-10 in an ex vivo re-stimulation system. Collectively, these findings demonstrate that the PCV2 truncated capsid subunit Fc-fusion protein can induce both cellular and humoral immune responses, and it displays great application potential.

Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus.

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two β-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

Sensitive lateral flow immunoassay for the detection of pork additives in raw and cooked meat products

In this study, we developed a lateral flow immunoassay (LFIA) for the detection of pork additives in meat products. LFIA of porcine immunoglobulins (IgG) as a molecular biomarker was carried out in a sandwich format for species identification. Gold nanoparticles as a nano-dispersed label were conjugated to secondary antibodies specific to anti-porcine IgG. The test system was characterized by high specificity, which was confirmed by the absence of cross-reactivity with any other species tested. A short technique of sample preparation was proposed aimed at the effective extraction of IgG from meat samples. The developed LFIA enabled the detection of a pork ingredient at a level as low as 0.063% (w/w) in raw meat within 35 min including sample preparation. A large panel of real meat samples was analyzed by the LFIA. The results showed that porcine IgG can be reliably recognized both in raw meat products and processed meat foodstuffs.

Smartphone-based competitive immunoassay for quantitative on-site detection of meat adulteration

Rapid, sensitive, and portable analytical methods for on-site inspection of food fraud are now an urgent requirement to ensure food quality and satisfy the ethnic considerations of consumers. Hence, for the first time, a colorimetric smartphone-based immunoassay was developed for the on-site detection of pork adulteration in meat. In detail, the immunoassay was based on a competitive strategy in which immobilized standard porcine IgG competed with the target porcine IgG extracted in a single step from meat samples. The parameters involved in each step of the immunoassay conception and the digital colorimetric detection were carefully investigated and optimized. Using polystyrene microplates as ready-to-use stable and portable immunoplatforms, TMB as chromogenic substrate, smartphone as signal readout, and Image J software for image processing; the developed immunoassay was able to detect as low as 0.01% of pork in meat mixtures in a total assay time of 30 min. The selectivity of the immunoassay was evaluated for different meat species, and it was shown to selectively respond only to pork. Furthermore, excellent stability of the prepared immunological platform was demonstrated under extreme temperature conditions (50 °C), which confirms its high portability potential for in situ quantification of pork, while being relatively cost effective and non-laborious. The developed method also provides great precision (RSD < 6%) and accuracy (relative error< 6%). Given the universal use of smartphones as portable and affordable devices, such format of immunoassay could be a promising approach for rapid and sensitive real-time monitoring of food fraud.

Lateral flow assay of methicillin-resistant Staphylococcus aureus using bacteriophage cellular wall-binding domain as recognition agent

As one of the most common and noticeable superbugs, methicillin-resistant Staphylococcus aureus (MRSA) has long been a major threat to public health. To meet the demand for effective diagnosis of MRSA-induced infection, it is urgent to establish rapid assay method for this type of pathogen. In this study, an aqueous soluble cellular wall-binding domain (CWBD) protein from bacteriophage P108 was obtained with a recombinant expression technique. It can act as a wide-spectrum binding agent for all MRSA strains and exclude the interference from methicillin-susceptible strains of Staphylococcus aureus and other species of bacteria. To establish a lateral flow assay (LFA) method for MRSA, CWBD-coupled time-resolved fluorescent microspheres (FMs) were used as signal probes for tracing MRSA, and a nitrocellulose membrane immobilized with porcine IgG was used to capture MRSA. With the LFA based on sandwich format, MRSA can be assayed within 10 min with a broad linear range of 6.6 × 102–6.6 × 107 CFU/mL. Its application potential has been demonstrated by assaying different types of bacteria-contaminated real samples. The results suggest that the LFA strip using recombinant CWBD as the recognition agent provides a rapid, portable, cost-effective approach for point-of-care testing of MRSA.

Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious virus that has led to enormous economic loss worldwide because of ineffective prevention and treatment. In view of their minimized size, high target specificity and affinity, nanobodies have been extensively investigated as diagnostic tools and treatments of many diseases. Previously, a PRRSV Nsp9-specific nanobody (Nb6) was identified as a PRRSV replication inhibitor. When it was fused with cell-penetrating peptide (CPP) TAT, Nb6-TAT could enter the cells for PRRSV suppression. However, delivery of molecules by CPP lack cell specificity and have a short duration of action. PRRSV has a tropism for monocyte/macrophage lineage, which expresses high levels of Fcγ receptors. Herein, we designed a nanobody containing porcine IgG Fc (Fcγ) to inhibit PRRSV replication in PRRSV permissive cells. Fcγ fused Nb6 chimeric antibody (Nb6-pFc) was assembled into a dimer with interchain disulfide bonds and expressed in a Pichia pastoris system. The results show that Nb6-pFc exhibits a well-binding ability to recombinant Nsp9 or PRRSV-encoded Nsp9 and that FcγR-mediated endocytosis of Nb6-pFc into porcine alveolar macrophages (PAM) was in a dose-dependent manner. Nb6-pFc can inhibit PRRSV infection efficiently not only by binding with Nsp9 but also by upregulating proinflammatory cytokine production in PAM. Together, this study proposes the design of a porcine IgG Fc-fused nanobody that can enter PRRSV susceptible PAM via FcγR-mediated endocytosis and inhibit PRRSV replication. This research reveals that nanobody-Fcγ chimeric antibodies might be effective for the control and prevention of monocyte/macrophage lineage susceptible pathogeneses.

Impact of Glycosylation and Species Origin on the Uptake and Permeation of IgGs through the Nasal Airway Mucosa.

Although we have recently reported the involvement of neonatal Fc receptor (FcRn) in intranasal transport, the transport mechanisms are far from being elucidated. Ex vivo porcine olfactory tissue, primary cells from porcine olfactory epithelium (OEPC) and the human cell line RPMI 2650 were used to evaluate the permeation of porcine and human IgG antibodies through the nasal mucosa. IgGs were used in their wild type and deglycosylated form to investigate the impact of glycosylation. Further, the expression of FcRn and Fc-gamma receptor (FCGR) and their interaction with IgG were analyzed. Comparable permeation rates for human and porcine IgG were observed in OEPC, which display the highest expression of FcRn. Only traces of porcine IgGs could be recovered at the basolateral compartment in ex vivo olfactory tissue, while human IgGs reached far higher levels. Deglycosylated human IgG showed significantly higher permeation in comparison to the wild type in RPMI 2650 and OEPC, but insignificantly elevated in the ex vivo model. An immunoprecipitation with porcine primary cells and tissue identified FCGR2 as a potential interaction partner in the nasal mucosa. Glycosylation sensitive receptors appear to be involved in the uptake, transport, but also degradation of therapeutic IgGs in the airway epithelial layer.

A sensitive colorimetric immunoassay based on poly(dopamine) modified magnetic nanoparticles for meat authentication

Magnetic nanoparticles as solid substrate and polydopamine as coating polymer were combined for the construction of a sensitive colorimetric immunoassay aimed at the quantification of pork associated with meat fraud. In this study, a competitive assay was conducted between purified IgGs and porcine IgGs covalently immobilized on magnetic nanoparticles, for the binding sites of peroxidase-labelled antibody. The recognition of porcine IgG was monitored by a simple colorimetric method in the presence of peroxidase substrate. The performed immunoassay was successfully employed for the screening of pork in adulterated meat, allowing low level of 0.01% to be detected in short time of analysis. The specificity of the immunoassay was tested against five different meat species (pork, lamb, turkey, chicken and beef) and it was found to be highly specific towards pork. Furthermore, the used magnetic probe has maintained a satisfactory stability enabling sensitive detection of pork within 20 days of storage. With the versatility and facile-preparation of polydopamine modified magnetic nanoparticles as well as the easy handling of the whole assay, this colorimetric method provides a specific and sensitive in-field-test for the detection of pork in adulterated meat and thus verifying halal meat authenticity.

Enzyme-Linked Immunosorbent Assay for Detection of Serum or Mucosal Isotype-Specific IgG and IgA Whole-Virus Antibody to Influenza A Virus in Swine.

Enzyme-linked immunosorbent assays can be used to detect isotype-specific anti-influenza antibodies in biological samples to characterize the porcine immune response to influenza A virus (IAV). The isotype antibody assay is based on an indirect ELISA using whole influenza virus as antigen and commercial antibodies directed against porcine IgG and IgA. Samples such as serum, nasal wash, and bronchoalveolar lavage fluid allow for evaluation of systemic, upper, and lower respiratory tract mucosal antibody responses, respectively. The isotype ELISA assay is performed in a 96-well format using IAV test antigen and anti-swine IgG or IgA detection antibodies conjugated to an enzyme that catalyze a color change reaction. The optical density of the sample is measured using an automated plate reader. The assay is useful to characterize the IgG or IgA immune response to challenge or vaccination against specific IAV isolates in different compartments of the immune system.

Antibiotic-affinity chromatographic test strip for quantitative analysis and antibiotic resistance testing of Staphylococcus aureus

Bacterial analysis and antibiotic resistance testing (ART) are of great significance in clinical diagnosis and therapy of bacterial infectious diseases. In this work, a portable antibiotic-affinity chromatographic test strip has been developed for rapid analysis of Staphylococcus aureus (S. aureus) and further applied for ART of this pathogen. Porcine IgG was immobilized on a nitrocellulose membrane for capturing S. aureus based on the selective binding capability of the Fc fragment of IgG toward protein A on the surface of the target bacteria. Fluorescent microspheres modified with teicoplanin (TEI) were applied as signal substances to trace S. aureus utilizing the hydrogen bond conjugation between this antibiotic and Gram-positive bacteria. S. aureus can be analyzed within the concentration range from 1.4 × 103 CFU mL−1 to 1.4 × 107 CFU mL−1. The recovery values for spiked samples were 93.3–110.0%. The obtained results of ART for penicillin, daptomycin, gentamicin, cefoxitin and clindamycin against S. aureus showed agreement with those of traditional broth dilution method. The procedures for bacterial analysis and ART can be accomplished within 20 and 110 min, respectively. The antibiotic-affinity chromatographic test strip showed great promise in point-of-care testing because of its ideal portability and rapidity.

Effect of Lyophilized Bovine Colostrum on Growth and Survival of Newborn Piglets From Local Breed, in Benin Republic

In Benin, the effect of supplementation of newborn piglets from local breed with lyophilized bovine colostrum was evaluated over a 49-d trail on the survival, the growth performances and the absorption of bovine and porcine immunoglobulin G (IgGs). Three groups of 24 piglets, stemming from five sows (Parity 2) belonging to a unique traditional farm, were respectively fed for the first 7 d of life with only sow colostrum (SC group n = 9), shea butter in addition to suckling (SCB group; n = 7) and a mixture of lyophilized bovine colostrum and shea butter in addition to suckling (BCB group; n = 8). Risk of diarrhea was not affected by the treatments, but BCB treatment tended to reduce it (P = 0.09). Diarrheal and healthy piglets had similar average levels of serum porcine IgG at 10 d (13.9 ± 1.6 mg/ml vs 14.7 ± 1.4 mg/ml, P &gt; 0.05). No bovine IgG was detected in the piglets serum at 10 d of age. Serum porcine IgG at 10 d was not affected by the treatments (P &gt; 0.05). However, bovine colostrum improved serum IgG at 49 d (P &lt; 0.05). No significant difference (P &gt; 0.05) was observed between the three groups for body weight and Average Daily Gain (ADG). By contrast, Relative Average Daily Gain (RADG) was affected by the treatments (P &lt; 0.05). The lyophilized bovine colostrum improved numerically ADG when compared to the piglets fed only with the colostrum from the sow and those fed also shea butter (41g vs 35 and 36 g/d for the first and second groups respectively). A higher RADG was obtained with the piglets from the BCB group (31 ± 1.6 g/d.kg vs 25.3 ± 1.6 g/d.kg and 24.5 ± 1.9 g/d.kg for the SC and SCB groups respectively). Lyophilized bovine colostrum improved RADG, active immunity at weaning and tended to reduce prevalence of diarrhea in newborn piglets.

Molecular recognition strategy for detection and antimicrobial susceptibility testing of Staphylococcus aureus by utilizing teicoplanin and porcine IgG as indicator molecules

Timely and accurate anti-infective therapy significantly benefits treatment of various life-threatening bacterial infectious diseases. Unfortunately, owing to the lack of selectivity for recognizing bacterial species, most of the reported phenotypic antimicrobial susceptibility testing protocols suffer from time-consuming bacterial isolation and identification. In this work, a highly-selective molecular recognition-based sandwich fluorimetric assay was developed to rapidly detect Staphylococcus aureus (S. aureus), and furtherly to assess its susceptibility to antibiotics. Immobilized porcine IgG was utilized to capture S. aureus through the selective interaction between Fc fragment of porcine IgG and protein A in the cell wall of S. aureus. Fluorescein isothiocyanate-labeled teicoplanin was adopted as the signal tracer utilizing the binding behavior between teicoplanin and d-Ala-d-Ala moieties in the peptidoglycans of Gram-positive bacterial cell wall. S. aureus could be detected within a wide linear rang of 1.0 × 103 to 1.0 × 107 CFU mL−1. Subsequently, this protocol utilizing porcine IgG and teicoplanin as the indicator molecules was utilized to assess the antimicrobial susceptibility of S. aureus after the bacteria were treated with antibiotics. The minimum inhibitory concentrations of penicillin, cefoxitin, clindamycin, trimethoprim/sulfamethoxazole and erythromycin were estimated to be >0.25,<4, <0.25, <2/38 and <0.5 μg mL−1, respectively. The whole process for antimicrobial susceptibility testing can be completed within 4 h since it avoided extra time-consuming isolation and identification of bacterial species.

Presence of Pig IgG and IgM in Sera Samples From Baboons After an Orthotopic Liver Xenotransplantation

IntroductionThe immunorejection in xenotransplantation has mostly been studied from the host's immune system activation point of view and there is very little information about the graft-vs-host reaction. ObjectivesTo validate an enzyme-linked immunosorbent assay (ELISA) test for porcine IgM and IgG quantitation, the assessment of porcine IgG and IgM in sera samples from baboons after liver orthotopic xenotransplantation or in human plasma after xenotransfusion through pig organs, and to assess the presence of porcine immunoglobulin in a baboon after plasmapheresis to a complete change of plasma after 4 passages through pig liver. Materials and MethodsTwo commercial ELISA kits for pig IgG and IgM quantitation were evaluated for cross reactivity with samples from baboons, Rhesus monkeys, squirrel monkeys, and humans. Then, samples from 18 baboons after orthotopic liver xenotransplantation were studied for porcine IgG and IgM. To understand the phenomenon, human plasma samples after xenotransfusion 1, 2, 3, or 4 times through liver or kidney were assessed for porcine IgG presence and finally, the porcine IgG were quantified in sera samples obtained during more than 4 years from a baboon after plasmapheresis with baboon plasma after xenotransfusion 4 times through a pig liver. ResultsPorcine IgG and IgM were found in samples from xenotransplanted baboon during all survival. The quantity of porcine IgG in plasma after xenotransfusion correlated with the number of passages through the pig liver, and the IgG were completely cleared from the baboon 16 days after plasmapheresis and complete substitution of plasma after 4 xenotransfusions through a pig liver.

Fluorescence Microsphere Immunoassay for Detection of Antibodies to Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus type 2 using Protein A, Protein G, and Protein A/G

The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) specific IgG antibodies by incorporation of non-species-specific conjugates Protein A, G, and A/G in place of the secondary antibody. A total of 205 serum samples obtained from pigs experimentally infected with PRRSV and/or PCV2 were tested. For the production of recombinant antigens, PRRSV nucleoprotein (N) and PCV2 capsid protein (CP) were expressed in Escherichia coli and purified on a nickel affinity column. The purified proteins were covalently coupled to carboxylated microsphere beads. Four target antigens were assembled into a single multiplex and tested against sera from swine infected or co-infected with PRRSV or PCV2. Protein A, G, and A/G have been tested in place of secondary conjugate porcine IgG. All conjugates were capable of detecting antibody and the detection of IgG responses against PRRSV and PCV2 antigen targets were varied with A> A/G> G.In the absence of species specific reagents the incorporation of Protein A, G, and A/G provide a suitable substitutes.